5-Nitropyrimidines

ABSTRACT

Novel pteridines of formula (I), ##SPC1## 
     wherein R is an optionally substituted phenoxyalkyl group, and R 1  and R 2  are the same or different and each is a lower alkyl group or R 1  and R 2 , together with the carbon atom in the pteridine ring structure, form a spirocycloalkyl ring system having 4 to 6 carbon atoms outside the pteridine ring structure, 
     And their method of preparation. 
     The above compounds have bacteriostatic activity.

The present invention relates to derivatives of pteridine, theirchemical synthesis and pharmaceutical formulations containing them. Thespecification also describes compositions and pharmaceuticalformulations comprising these pteridines in combinations which areuseful in the treatment of microbial infections.

It is already established that the compounds2-amino-4-hydroxy-6-hydroxymethyl-7,7-dimethyl-7,8-dihydropteridine and2-amino-4-hydroxy-6-methyl-7,7-dimethyl-7,8-dihydropteridine or theirtautomers or pharmaceutically acceptable salts thereof, havebacteriostatic activity, being particularly effective against Cl.perfringens and Derm. dermatonomous, as disclosed in the specificationsof British Pat. No. 1303171 and application Ser. No. 36289/70 (BelgianPat. No. 770,577).

It has now been found that the novel pteridines represented by thefollowing formula (I) or their tautomers or pharmaceutically acceptablesalts thereof, ##SPC2##

Wherein R is an optionally substituted phenoxyalkyl group, and R¹ and R²are the same or different and each is a lower alkyl group or R¹ and R²,together with the carbon atom in the pteridine ring structure, form aspirocycloalkyl ring system having 4 to 6 carbon atoms outside thepteridine ring structure, are also useful as antagonists of microbialmetabolism. Furthermore these compounds may be useful as intermediatesfor the synthesis of the afore-mentioned 6-hydroxymethyl and 6-methylsubstituted pteridines.

Although the phenoxyalkyl group is preferably unsubstituted and is mostpreferably a phenoxymethyl group, it may be substituted with one ormore, advantageously one or two, alkoxy, amino, lower alkyl or hydroxylgroups or halogen atoms, in particular with a substitution in the paraposition. Compounds substituted with a methyl or methoxy group or achlorine atom are of particular interest. Furthermore R¹ and R² arepreferably lower alkyl groups and are most preferably the same and bothmethyl groups or ethyl groups. The compound2-amino-4-hydroxy-6-phenoxymethyl-7,7-dimethyl-7,8-dihydropteridine ismost preferred.

As used herein and throughout the specification, the terms "lower alkylgroup" or "alkoxy group" refer to groups containing a straight orbranched chain alkyl group having 1 to 4 carbon atoms.

According to the present invention, therefore, there are provided in oneaspect the novel compounds of formula (I).

The above compounds and their salts inhibit one of the enzymes involvedin the biosynthesis of dihydrofolic acid, namelyhydroxymethyldihydropteridine pyrophosphokinase, which is essential tothe growth of microorganisms, for instance bacteria. They can thus beused in in vitro pharmacological investigations in clinical anddiagnostic tests establishing, for instance, the properties of bacteria.When used as bacteriostats they may be present in a concentration of 50to 500, in particular 110 to 180 mg of base/ml of the solution in whichthe organism grows in the absence of a compound. A further use of thecompounds, when in solution, is in the treatment of wounds, for exampleafter surgery, to prevent the growth of bacteria. Moreover the compoundsof formula (I) and their salts manifest unexpectedly low toxicity inmammals or birds e.g. poultry, which makes them particularly suitablefor application against microbial infections in such hosts undercircumstances hereinbelow described.

Tetrahydrofolate co-factors are essential metabolites in all cells forthe biosynthesis of purines, thymidylic acid, serine and several otherbiologically important compounds. Most of these co-factors areone-carbon adducts of tetrahydrofolic acid. The ultimate source of thesefor higher animals and man is food, containing preformed folates usuallyin the form of vitamins.

In microorganisms, the co-factors are synthesised from simplerchemicals. Generally the bio-synthetic process first provides`dihydropteridine` (Pt), i.e.2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine (HMPt)pyrophosphate ester, from its immediate precursor HMPt in the presenceof the enzyme hydroxymethyldihydropteridine pyrophosphokinase (HMPPS).Pt then condenses with p-aminobenzoic acid (pAB) in the presence of theenzyme dihydropteroate synthetase to form dihydropteroic acid (DPtA).This intermediate further condenses with a glutamate to formdihydrofolic acid (DFA or `folate`) which is then enzymatically reducedto provide the essential tetrahydrofolate in, for instance, bacteria andother micro-organisms.

The provision of the `folate` from the basic building blocks, i.e.pteridine, pAB, and glutamate, and the further conversion of this intothe tetrahydrofolate is known to be inhibited in two different ways. Forinstance sulphonamides displace pAB in the above reaction scheme.Because of their close structural resemblance to pAB, sulphonamides orsimilar other `competitors` enter the biosynthesis and prevent theformation of DPtA, and of DFA, and are therefore antimetabolites for themetabolite pAB. It is also known that compounds which are `inhibitors`of the enzyme dihydrofolic acid reductase block the synthetic stepleading to tetrahydrofolate. A considerable number of pyrimidinederivatives show substantial anti-microbial properties on the basis ofsuch blockage.

It was established later that such inhibitors may act synergisticallywith sulphonamides, i.e. there can be a sequential double blockade and astrong mutual potentiation of the anti-bacterial effects of the twomaterials. The range of anti-microbial action exerted by suchcombinations is considerably wider than that expected from the activityof either drug, and organisms which are only marginally sensitive to theindividual agents become very sensitive to the combinations.

It was also suggested hypothetically that antimetabolites to Pt couldinhibit the biosynthesis of DPtA (and DFA) (cf. Hitchings and BurchallAdvances in Enzymology, 27, 417-468 (1965)) but compounds so far testedfor the purpose have been disappointing, being either inactive or tootoxic or sometimes both (cf. the compounds described in British Pat.Nos. 981,506 and 987,916).

It has been established that, for antimicrobial purposes, it is aprerequisite for the effective antagonism of Pt that the compound shouldbe an inhibitor of HMPPS without also acting as an antimetabolite to thedihydropteridine that serves as a cofactor for the hydroxylation ofphenylalanine and tyrosine, precursors of the catecholamines, such asnorepinephrine, that have important actions as regulators ofcardiovascular systems. Such an antimetabolic effect could lead toprohibitive toxicity to avian or mammalian species, which are normallythe hosts infected with the microbes.

It has now been found that the compounds of formula (I) and their saltsfulfil the above requirements i.e. inhibition of HMPPS combined with lowtoxicity to host species, as demonstrated for instance in chicks andrats. These compounds not only inhibit the growth of microorganisms ontheir own, albeit to a limited extent with certain bacteria, such asStaphylococcus aureus, Streptococcus pyogenes, Streptococcus faecalis,Escherichia coli, Salmonella typhi, Proteus vulgaris, Pseudomonasaerugenosa, Pasteurella multocida among others, but have been found toact with a most remarkable synergistic effect when combined with acompetitor of p-aminobenzoic acid, i.e. sulphonamides and similarcompounds, or with selective inhibitors of dihydrofolic reductase, i.e.pyrimidines and related compounds, or with a combination of both ofthese types of anti-microbial agents. This potentiating effect of thecompounds of formula (I) is the subject of co-pending cognate Britishpatent application Ser. No. 36774/71.

In that application there is described and claimed a composition fortesting or treating microbial systems or infections, comprising aneffective potentiating amount of a compound of formula (I) incombination with an effective amount of a competitor or inhibitor, orboth, as herein defined.

The microbial infections against which these combinations are effectiveare protozoal or bacteria infections caused by those microorganismswhich synthesise at least a substantial part of their tetrahydrofolatecofactor requirements. More specifically these infecting microorganismsare those which adequately absorb the pharmaceutical combinationsdisclosed herein and further are those in which these combinations havea synergistic effect in interfering with the de novo synthesis of therequired tetrahydrofolate co-factors. For example, the compositionsdescribed have been found to be useful in the treatment of infectionscaused by Staphylococcus aureus, Pseudomonas aerugenosa and Pasteurellamultocida.

It has been found specifically that, when compounds of formula (I) arecombined with an amount of the competitor and/or the inhibitor which isnot ordinarily sufficient to be effective as an antimicrobial agent inits own right, the combination of a compound of formula (I) with thisnormally ineffective amount of the competitor and/or the inhibitorprovides a composition which in totality acts as an effectiveantimicrobial agent. This is especially notable when the amount of thecompound of formula (I) is so low that it has substantially no microbialeffect at the particular level, yet in the combination the potentiationis marked, in some instances very marked. Thus by using an effectivepotentiating amount of a compound of formula (I) together with thecompetitor and/or the inhibitor, it is now possible to reducesignificantly the amount of the competitor and/or the inhibitor requiredto inhibit the growth of these bacteria.

In accordance with the above therefore, the term "an effective amount"used in conjunction with the terms a dihydrofolic reductase `inhibitor`and a para-aminobenzoic acid `competitor` means either (a) an amount ofthe `inhibitor` or `competitor` which is effective to a degree as anantimicrobial agent in its own right but which is potentiated by the useof a compound of formula (I) or (b) an amount of the `inhibitor` or`competitor` which is ineffective as an antimicrobial agent but whichwhen combined with a compound of formula (I) provides a compositionwhich is an effective antimicrobial agent. An "effective potentiatingamount" means an amount of the compound of formula (I) which increasesthe activity of an inhibitor and/or a competitor so as to provide animproved or adequate effectiveness for the whole combination.

It should be emphasised that the inhibition of the biosyntheticprocesses by such means could be termed as competitive antagonism in allthree instances, and there might be potentiation between all three typesof agents. The terms `inhibitor`, `competitor`, and `potentiation` by acompound of formula (I) are arbitrary and should only serve asconvenient names for the appropriate type of components in combinationproducts described and claimed in the specification of theafore-mentioned cognate application.

The inhibiting activity against HMPPS of a selected compound of formula(I) can, for instance, be tested by monitoring the transfer of theterminal phosphate of adenosine triphosphate ATP-γ-P³² to`dihydropteridine`. It was found that the concentrations required for50% inhibition of the formation of Pt (IC₅₀) in such tests are wellcorrelated and within the margin of error obtained by other relevanttests in this respect, which measure the inhibition of either of the twoenzymes involved in the formation of HMPt and DPtA. Such inhibition may,for instance, be easily and simply carried out by incubating an extractof E. coli with pAB-7-C¹⁴, ATP, Mg and `dihydropteridine`. The formationof the dihydropteroate-C¹⁴ can be quantitatively assayed afterseparating the unreacted pAB substrate, for instance by chromatography.It has been found that compounds possessing in such tests an IC₅₀ valueof about 100μM or less, usually below 50μM represent compounds exertinga useful potentiating effect, provided their toxicity in the appropriatevertebrates is acceptable. Preferably the value is 25μM or less, such asin the range between 2 to 12μM. Generally a value below 7μM isdesirable.

As explained above, for the purpose disclosed it is essential that thecompound of formula (I) should not have a prohibitive toxicity to themammalian or avian hosts' cardiovascular systems. While low toxicity istherefore an essential requirement, a therapeutic index incorporatesboth the activity and toxicity values pertinent to the presentdisclosure and could be used with advantage for the selection ofpotentiating compounds of formula (I).

The therapeutic index is defined as the ratio of the maximum tolerateddose to the minimum effective dose and in most cases is preferablygreater than 10, suitably at least 5 and in exceptional circumstances atleast about 3 for humans, but possibly as low as 2 for animals.

Although the art is aware of many compounds which are known competitorsof para-aminobenzoic acid and are antimicrobials, the sulphur compoundswhich are disclosed as antimicrobial agents from the top of page 994 topage 1007 of the Merck Index, 8th Edition, 1968 are presented by way ofexample only.

Of the known compounds which are competitors, the following sulphonamidecompounds (or pharmaceutically acceptable salts thereof) are preferredfor the purpose described:

sulphanilamide, sulphadiazine, sulphamethisazole, sulphamethizole,sulphapyridine, sulphathiazole, sulphamerazine, sulphamethazine,sulphisoxazole, sulphadoxine, sulphasomidine, sulphachlorpyridazine,2-(p-aminobenzene)sulphonamido-3-methoxypyrazine(Kelfizina),α-amino-p-toluenesulphonamide, 5-sulphanilamido-2,4-dimethyl pyrimidine,4-(N'-acetyl sulphanilamido)-5,6-dimethoxy pyrimidine,3-sulphanilamido-4,5-dimethyl isoxazole,4-sulphanilamido-5-methoxy-6-decyloxy pyrimidine, sulphamonomethoxine,4-p-(8-hydroxy-quinilinyl-4-azo)-phenyl sulphanilamido-5,6 -dimethoxypyrimidine, sulphadimethoxine, sulphamethoxazole, sulphaquinoxaline, andp-(2 methyl-8-hydroxy-quinolinyl-(5)-azo)phenylsulphanilamido-5,6-dimethoxy pyrimidine. Examples of a non-sulphonamidetype of competitor are p-amino salicylic acid (PAS) andp,p'-diaminodiphenylsulphone.

Similarly, although many compounds are known which inhibit dihydrofolicreductase and act as antimicrobial agents, the compounds disclosed inthe following patents are presented by way of example of compoundssuitable for use for the purpose disclosed:

U.S. Pat. Nos. 2,658,897; 2,767,183; 3,021,332; 2,937,284; 3,322,765;2,909,522; 2,624,732; 2,579,259; 2,945,859; 2,576,939; 2,926,166;2,697,710; 2,749,345; and 2,749,344.

The following inhibitors (or pharmaceutically acceptable salts thereof)are preferred for the combinations described, however:

2,4-diamino-6-ethyl-5-p-chlorophenylpyrimidine (pyrimethamine),2,4-diamino-5-(3'4',5'-trimethoxybenzyl)-pyrimidine (trimethoprim),2,4-diamino-5-(3'4'-dimethoxybenzyl) pyrimidine (diaveridine),2,4-diamino-5-(2'-isopropyl-4'-chlorophenoxy) pyrimidine,2,4-diamino-5-methyl-6-sec-butylpyrido (2,3-d) pyrimidine,2,4-diamino-5-methyl-6-benzylpyrido(2,3-d) pyrimidine,2,4-diamino-6-benzylpyrido(2,3-d) pyrimidine,2,4-diamino-5-6-trimethylenequinazoline,

2,4-diamino-5,6-tetramethylenequinazoline,2,4-diamino-5-(2',4'5'-trimethoxybenzyl) pyrimidine,2,4-diamino-5-(2'-ethyl-4',5-dimethoxybenzyl) pyrimidine,2,4-diamino-5-(2'-methyl-4',5'-dimethoxybenzyl) pyrimidine.

However, the most preferred combinations include those combining acompound of formula (I), especially that wherein R is a phenoxymethylgroup and R¹ and R² are both methyl groups, with sulphadiazine,sulphamethoxazole, sulphadoxine or sulphaquinoxaline as competitors, orwith trimethoprim, diaveridine or pyrimethamine as inhibitors. In viewof possible synergistic advantages of using certain competitors andinhibitors in combination against particular diseases, and thepotentiating effect of compounds of formula (I) on both of these typesof antibacterial compounds, it has been preferred to formulate triplecombinations, comprising a compound of formula (I) with one of theabove-mentioned preferred competitors, and one of such inhibitors. Forexample, combinations of sulphadiazine/trimethoprim,sulphamethoxazole/trimethoprim, sulphadoxine/trimethoprim orsulphaquinoxaline/diaveridine, each together with a compound of formula(I), give improved effectiveness when compared with the components aloneor with pairs of them.

The compounds of formula (I) either alone or together with thecompetitor and/or the inhibitor, may be presented in association with acarrier in pharmaceutical formulations suitable for parenteral, topical,rectal or oral administration. The formulations for oral or rectaladministration are advantageously presented in discrete units, such astablets, capsules, cachets, ampoules or suppositories, each containing apredetermined amount of each compound, but may also be presented as apowder, as granules, as a solution or suspension in an aqueous ornon-aqueous liquid, or as an ointment or paste for topicaladministration. For parenteral use, the formulations incorporating anaqueous or non-aqueous liquid carrier must be sterile and be presentedin sealed containers. The formulations may be made by any of the knownmethods and may include one or more of the following accessoryingredients:

diluents, solutes to render the solution isotonic with the blood,buffers, flavouring, binding, dispersing, surface-active, thickening,lubricating and coating materials, preservatives, bacteriostats,antioxidants, suppository and ointment bases, and any other acceptableexcipients.

In another aspect of the present invention, therefore, there is provideda pharmaceutical formulation comprising a compound of formula (I) incombination with a pharmaceutically acceptable carrier. In yet anotheraspect the present invention provides a method of making apharmaceutical formulation by admixing the compound of formula (I) witha carrier by known techniques. The specification of the afore-mentionedcognate application further discloses and claims a pharmaceuticalformulation comprising a composition, as hereinbefore defined, togetherwith a carrier, and its method of preparation, by admixing thecomposition with the carrier by known, techniques.

Formulations containing the compound of formula (I) in association witha competitor or an inhibitor may also be presented in the form of a kit,which comprises separately packaged units or dosages of these componentswith instructions for use in a combined form. The instructions may alsospecify the manner of administration and indications for which theformula is suitable.

The compounds of formula (I), either for use alone or in associationwith a competitor and/or inhibitor, and also the competitors andinhibitors, may be presented in the form of their pharmaceuticallyacceptable salts of a mineral or organic acid, for example hydrochloricacid, hydrobromic acid, sulphuric acid, acetic acid, citric acid,tartaric acid, lactic acid, maleic acid or salicylic acid, or,especially for the sulphonamide competitor, of a base, such as sodiumhydroxide, potassium hydroxide, tetramethyl ammonium hydroxide orammonia.

The ratios in which the therapeutically active compounds of formula (I)are utilized in the compositions described in this specification can bevaried between wide limits. Depending on the nature and circumstance ofuse, the compositions may contain the compound of formula (I) with thecompetitor and/or the inhibitor in appropriate proportions and dosages.For instance, in cases of uses in vivo it is often desirable to maintaina certain proportion of components in the blood serum or tissue fluids,preferably for a prolonged period. Depending on the various absorption,discharge or decomposition rates of the components, the initialquantities and proportions of the ingredients of the formulation can bedifferent from that aimed at in the tissues in vivo. The formulationsand dosages recommended for the general treatment of a particular humanor animal disease must be adjusted according to the particularrequirements of the recipients of the disease, the known activities ofthe competitor or inhibitor component against the causative organism,the half life and the toxicity of the components in vivo, and otherpractical requirements.

For example the composition or pharmaceutical formulation may containfrom about 1 to 30 parts by weight, preferably 5 to 15 parts of thecompound of formula (I), or an equivalent amount of a salt thereof, and1 to 30 parts, preferably 5 to 15 parts, of a competitor, or anequivalent amount of a salt thereof, and/or one part of an inhibitor, oran equivalent amount of a salt thereof.

Dosage will vary depending upon the infecting organism but underordinary circumstances up to about 60 mg/kg each of a compound offormula (I) and competitor, and up to about 7.5 mg/kg of inhibitor, incombination, can be administered daily in several doses. The compositionor pharmaceutical formulation can be administered to human patients inunit dosage forms which contain up to 750 mg of the compound of formula(I), and up to 750 mg of the competitor and/or up to 25 mg of theinhibitor. Preferably for adult dosages the amount of the compound offormula (I) would be about 200 mg, that of the competitor about 200 mgand/or that of the inhibitor about 25 mg.

The pharmaceutical formulation comprising the compound of formula (I) incombination with the competitor and/or the inhibitor is also usable insolution for irrigating wounds, for example after surgery, so as toprevent the growth of bacteria. For example, an antibacterial solutionhaving the following preferred concentration of components may be used:

1-30 mg/ml of the compound of formula (I), 1-30 mg/ml of the competitorand/or 0.03-1 mg/ml of the inhibitor, in a pharmaceutically acceptablesolvent, suitable for external use.

The potentiating effect of compounds of formula (I) can be demonstratedand utilized in vitro relatively easily for research and practicalpurposes. Such possibilities include diagnosis and the identification ofthe bacterial flora of individuals and the consequential selection ofclinical treatment schedules.

The various combinations can be incorporated in porous discs (such asfilter paper discs) or in Agar Nutrient or other media for bacterialgrowth for determining susceptibility. Those articles incorporating thecompound of formula (I) with a competitor and/or an inhibitor compoundmay be distributed or sold to doctors, hospitals and clinics for theabove purposes. A typical testing disc may be impregnated with asolution containing 5 to 50 μg/ml of a para-aminobenzoic acidcompetitor, 0.5 to 5 μg/ml of a dihydrofolic reductase inhibitor, andabout 10 to 100 μg/ml of a compound of formula (I) in a mediumcomprising a mixture of an aqueous infusion and papain digest of horsemuscle.

Furthermore, such pharmacological tests involving potentiatedcompetitors or inhibitors may also be useful for the characterisation ofbacteria according to their sensitivity and to their particularresistance for instance to a competitor when used alone, and suchinvestigations involving a variety of formulations as described hereinalso form the basis of determining the compositions of selectedformulations for general treatment purposes. The toxicity of compoundsof formula (I) is generally considerably lower than that of thecompetitors or inhibitors commonly used, which may enable the clinicianto maintain or increase the effectiveness of the antibacterial activityof the formulation with a concurrent increase of the therapeutic ratioor decrease in the toxic or side-effects of the medicament.

In addition to the above, compounds of formula (I) have been found topotentiate the activity of the aforementioned competitors and/orinhibitors against infections with microorganisms in domestic animals,including poultry, for example against Pasteurella multocida butespecially against the protozoal disease coccidiosis. Such tripleformulations comprising a compound of formula (I) together with acompound such as sulphaquinoxaline and an inhibitor such as diaveridineare effective in lower concentrations than the competitor or inhibitorcomponents alone and possess an enhanced activity, being effectiveagainst all relevant Eimeria species causing this disease in poultry.

The compounds of formula (I) may be prepared by the reductivecyclisation of a compound of formula (II), ##SPC3##

wherein R, R¹ and R² are as defined above and Z is a ketonic oxygen atomor a protecting group therefor, such as a semicarbazone group or anoxime group, prepared according to the procedures disclosed byPfleiderer and Zondler (Chem. Ber. 99, 3008 (1966)) and thespecifications of British Pat. No. 1303171 and of co-pending Britishpatent application Ser. No. 36289/70 (Belgian Pat. No. 770,577)respectively.

The method described in British patent application No. 36289/70,however, is particularly preferred.

In this method, a compound

    R.sup.1 R.sup.2 C = CHR                                    (VI),

wherein R, R¹ and R² are as defined above, undergoes an additionreaction with a nitrosyl halide, prepared in situ, and the resultingnitrosohalide (V) is converted to the oxime (IV) by reaction withammonia solution. Reacting the oxime (IV) with a2-amino-4-halogeno-6-hydroxy-5-nitropyrimidine (III) provides thepyrimidine ketoxime (II) which is then reductively cyclised to give thpteridine (I), as shown in the following sequence. ##SPC4##

The compound (VI) may in turn be prepared by reaction of thecorresponding halide of formula (VII), wherein n is an integer from 1 to4, with an alkali metal phenoxide, optionally substituted in the phenylring, in the presence of a polar solvent. The halide (VII) may beprepared from the halogentaion corresponding primary alcohol (VIII) byhalogenation with an appropriate agent, such as a phosphorus trichlorideor thionyl chloride. The alcohol (VIII) may itself be obtained byreduction of a carboxylic acid or ester derivative, e.g. (IX), with apowerful reducing agent, such as lithium aluminium hydride or sodiumdihydrobisethoxymethoxy aluminate. Alternatively the tertiary alcohol,e.g. (X), resulting from the reaction of a ketone, e.g. (XI), with anallylic Grignard reagent, may be halogenated to produce the halide(VII). The above sequence is illustrated below for the preparation of anoptionally substituted phenoxymethyl derivative, but the higherhomologues may be prepared analogously. ##EQU1##

It will be seen from the above that in the preparation of compounds offormula (I) wherein the phenoxyalkyl group is substituted ashereinbefore defined, the phenyl ring in the alkali metal phenoxideshould already contain these substituents in the desired position. If acompound having R¹ and R² as different substituents is required, thenitrosohalide (V) will contain a racemic mixture of two stereoisomers,in view of the asymmetric carbon atom present. Separation of theseisomers by conventional techniques well known in the art mayadvantageously be effected at this stage prior to reaction with theammonia solution.

According to the present invention in further aspects there are alsoprovided:

1. The methods described herein for preparing any of the compounds offormula (I), comprising effecting reductive cyclisation of the compoundsof formula (II).

2. the methods described herein for preparing any of the compounds (II),wherein Z is an oxime group, from (IV), (IV) from (V) or (VI) and (VI)from (VII).

3. compounds of formula (I), (II), wherein Z is an oxime group, (IV) and(V), whenever prepared by a method as defined under (1) or (2).

4. As novel compounds of value as chemical intermediates: compounds offormula (II), (IV) and (V).

5. a pharmaceutical formulation comprising a compound of formula (I) ora salt thereof in combination with a pharmaceutically acceptablecarrier, whenever prepared by the method herein described.

The following examples illustrate the invention but are in no wayintended to limit the scope of the invention. Temperatures are indegrees Celsius.

EXAMPLE A2-Amino-4-hydroxy-6-phenoxymethyl-7,7-dimethyl-7,8-dihydropteridine (I;R= CH₂ OC₆ H₅, R¹ =R² =CH₃) i. 1-phenoxy-3-methylbut-2-ene (VI; R=CH₂ OC₆H₅,R¹ =R² =CH₃)

A mixture of phenol (18.8 g) and sodium hydroxide (8 g) was stirred at60°-70° in dimethylformamide (162 ml) and water (18 ml) while1-bromo-3-methylbut-2-ene (VII, R¹ =R² =CH₃, Hal=Br) (29.8 g) was addeddropwise over 11/2 h. The mixture was stirred at 60° for 4 h after theaddition was complete. It was then poured into water (800 ml) andextracted with ether (3 × 300 ml). The ether extract was washed with 10%sodium hydroxide (200 ml) and then with water (3 × 200 ml) before dryingover anhydrous sodium sulphate. The solvent was removed in vacuo, andthe residue distilled to give 1-phenoxy-3-methylbut-2-ene (2.4 g, yield75%) as a clear liquid b.p. 116°-118°/18 mm. Hg.

ii. 2-Chloro-4-phenoxy-2-methyl-3-nitrosobutane (V; R = CH₂ OC₆ H₅, R¹ =R² = CH₃)

Concentrated hydrochloric acid (7.8 ml) was added dropwise over 1 h to amixture of 1-phenoxy-3-methylbut-2-ene (VI) (12.6 g) and amyl nitrite(11.7 ml) in glacial acetic acid (15.6 ml) at 0° (ice-salt bath). Awhite precipitate separated and this was filtered off and dried to givethe mitrosachloride (V) (12.8 g, yield 73%) as colourless crystals, m.p.134°. The nitrosochloride can be recrystallised from acetone but this isnot necessary.

iii. 3-Amino-1-phenoxy-3-methylbutan-2-one oxime hydrochloride (IV; R =CH₂ OC₆ H₅, R¹ = R² = CH₃)

A suspension of 2-chloro-4-phenoxy-2-methyl-3-nitrosobutane (V) (17.2 g)in methanol (approx. 300 ml) was refluxed while ammonia gas was bubbledthrough the suspension. Methanol was added from time to time to keep thevolume of the solution approximately constant. After 6 h the solutionwas evaporated, and the residue after washing with acetone gave theketoxime hydrochloride (IV) (12.1 g, yield 65%) as colourless crystals,m.p. 238°-240° (decomp.).

iv.2-Amino-4-hydroxy-6-[2-hydroxyimino-3-phenoxy-1,1-dimethylpropylamino]-5-nitropyrimidine(II; R = CH₂ OC₆ H₅, R¹ = R² = CH₃)

A mixture of 2-amino-4-chloro-6-hydroxy-5-nitropyrimidine (III) (Hal =Cl) (4.8 g), 3-amino-1-phenoxy-3-methylbutan-2-one oxime hydrochloride(IV) (6.12 g) and dry triethylamine (5.45 g) in dry ethanol (50 ml) wererefluxed for 2 h. The mixture was cooled, water (150 ml) was added, andthe resulting precipitate was filtered off and dried. Recrystallisationfrom water gave the nitropyrimidine oxime (II) (5.45 g, yield 57%) aspale yellow plates, m.p. 210° (decomp.).

v. 2-Amino-4-hydroxy-6-phenoxymethyl-7,7-dimethyl-7,8-dihydropteridine(I; R = CH₂ OC₆ H₅, R¹ = R² = CH₃)

A solution of2-amino-4-hydroxy-6-(2-hydroxyimino-3-phenoxy-1,1-dimethylpropylamino)-5-nitropyrimidine(II) (3.6 g) in water (100 ml) and 2M sodium hydroxide (20 ml) waswarmed on a steam bath. Sodium dithionite (6.3 g) was added portionwiseuntil an almost colourless solution was obtained. Glacial acetic acidwas added to the solution to adjust to pH 4, and the mixture was heatedfor 1 h. The solution was cooled and the dihydropteridine (I) (2.0 g,yield 67%) was collected by filtration, m.p. >260° (decomp.).

EXAMPLE B 2-Amino-4-hydroxy-6-(p-methoxy)phenoxymethyl-7,7-dimethyl-7,8-dihydropteridine I; R = CH₂ OC₆ H₄.OCH₃, R¹ = R² = CH₃) i.1-p-methoxy)phenoxy-3-methylbut-2-ene (VI; R = CH₂ OC₆ H₄ .OCH₃, R¹ = R²= CH₃)

A mixture of p-methoxyphenol and sodium hydroxide was stirred withdimethylformamide and water while 1-bromo-3-methylbut-2-ene(VII; R¹ = R²= CH₃, Hal = Br) was added dropwise. The mixture was stirred, pouredinto water and extracted with ether. The ether extract was washed with10% sodium hydroxide and then with water before drying over anhydroussodium sulphate. The solvent was removed in vacuo, and the residuedistilled to 1-(p-methoxy)phenoxy-3-methyl-but-2-ene (VI).

ii. 2-Chloro-4-(p-methoxy)phenoxy-2-methyl-3-nitrosobutane (V; R = CH₂OC₆ H₄ .OCH₃, R¹ = R² = CH₃)

Concentrated hydrochloric acid (8.1 ml) was added dropwise over 10minutes to a mixture of 1-(p-methoxy)phenoxy-3-methylbut-2-ene (VI ; R =CH₂ OC₆ H₄ .OCH₃, R¹ = R² = CH₃) (16.05 g) and amyl nitrite (12.1 ml) inglacial acetic acid (16.2 ml) at -5° to 0° (ice-salt bath). After afurther 1 hour at -5° to 0°, the mixture was filtered. The solid soobtained was washed with acetone and recrystallised from acetone to givethe nitrosochloride (V) (4.87 g, yield 22%) as pale yellow crystals,m.p. 123° (decomp.).

iii. 3-Amino-1-(p-methoxy)phenoxy-3-methylbutan-2-one oximehydrochloride (IV; R = CH² OC₆ H₄ .OCH₃, R¹ = R² = CH₃)

A suspension of 2-chloro-4-(p-methoxy)phenoxy-2-methyl-3-nitrosobutane(V) (4.803 g) in dry methanol (80 ml) was heated to reflux while ammoniagas was passed through until all the solid dissolved (3.5 h). Filtrationand removal of solvents gave an off-white solid, which was largely freedof impurities by washing with benzene. Two recrystallisations fromisopropanol (a small inorganic residue remained) gave the ketoximehydrochloride (IV) (2.77 g, yield 53%) as colourless crystals, m.p.219°-220° (decomp.).

iv.2-Amino-4-hydroxy-6-[2-hydroxyimino-3-(p-methoxy)phenoxy-1,1-dimethylpropylamino]-5-nitropyrimidine(II; R = CH₂ OC₆ H₄.OCH₃, R¹ = R² = CH₃)

A mixture of 3-amino-1-(p-methoxy)phenoxy-3-methylbutan-2-one oximehydrochloride (IV) (2.748 g),2-amino-4-chloro-6-hydroxy-5-nitropyrimidine (III) (Hal = Cl) (1.906 g)and dry triethylamine (4.1 g) was heated to reflux in dry ethanol (120ml) for 17 hours. 120 ml water was added and the mixture was heated toeffect solution, filtered and cooled. The filtrate on removal of somesolvents under reduced pressure deposited the nitropyrimidine oxime (II)(1.77 g) as a tan solid, m.p. >250° (decomp.).

v.2-Amino-4-hydroxy-6-(p-methoxy)phenoxymethyl-7,7-dimethyl-7,8-dihydropteridine(I; R = CH₂ OC₆ H₄ .OCH₃, R¹ = R² = CH₃)

A solution of2-amino-4-hydroxy-6-[2-hydroxyimino-3-(p-methoxy)phenoxy-1,1-dimethylpropylamino]-5-nitropyrimidine(II) (1.60 g) in warm 1M sodium hydroxide (80 ml) was treated during 25minutes with sodium dithionite (8 g) in portions until no red colourremained. A pink solid was removed and the filtrate was acidified withglacial acetic acid. Most of the solvents were removed under reducedpressure. Treatment with methanol and filtration, concentration of thefiltrate and repetition gave an alcoholic concentrate from which thedihydropteridine (I) separated as pale needles.

EXAMPLE C2-Amino-4-hydroxy-6-(p-methoxy)phenoxymethyl-7,7-dimethyl-7,8-dihydropteridine(I; R = CH₂ OC₆ H₄.OCH₃, R¹ = R² = CH₃) i.1-(p-methoxy)phenoxy-3-methylbut-2-ene (VI; R = CH₂ OC₆ H₄ OCH₃, R¹ = R²= CH₃)

1-Bromo-3-methyl-but-2-ene (VII; R¹ = R² = CH₃) was added dropwise to amixture of p-methoxyphenol and sodium hydroxide in dimethylformamide andwater and the 1-(p-methoxy)phenoxy-3-methylbut-2-ene (VI) extracted asdescribed under Example B(i).

ii. 2-Chloro-4-(p-methoxy)phenoxy-2-methyl-3-nitrosobutane (V; R = CH₂OC₆ H₄.OCH₃, R¹ = R² = CH₃)

Concentrated hydrochloric acid (7.8 ml) was added dropwise over 1 hourto a mixture of 1-(p-methoxy)phenoxy-3-methylbut-2-ene (VI; R = CH₂ OC₆H₄.OCH₃, R¹ = R² = CH₃) (12.0 g) and amyl nitrite (11.7 ml) in glacialacetic acid (15.6 ml) at 0°C (ice-salt bath). A white precipitateseparated and this was filtered off and dried to give thenitrosochloride (V) (8.0 g, yield 50%) as colourless crystals, m.p.137°.

iii. 3-Amino-1-(p-methoxy)phenoxy-3-metylbutan-2-one oxime hydrochloride(IV; R = CH₂ OC₆ H₄.OCH₃, R¹ =R² =CH₃)

A suspension of 2-chloro-4-(p-methoxy)phenoxy-2-methyl-3-nitrosobutane(V) (4.0 g) in methanol (approx. 80 ml) was refluxed while ammonia gaswas bubbled through the suspension. Methanol was added occasionally tokeep the volume of the solution approximately constant. After 6 hoursthe solution was evaporated and the residue, after washings withacetone, gave the ketoxime hydrochloride (IV) (3.2 g, yield 75%) ascolourless crystals, m.p. 225°.

iv.2-Amino-4-hydroxy-6-[2-hydroxyimino-3-(p-methoxy)phenoxy-1,1-dimethyl-propylamino]-5-nitropyrimidine(II; R = CH₂ OC₆ H₄.OCH₃, R¹ = R² = CH₃)

A mixture of 2-amino-4-chloro-6-hydroxy-5-nitropyrimidine (III) (Hal =Cl) (1.53 g), 3-amino-1-(p-methoxy)phenoxy-3-methylbutan-2-one oximehydrochloride (IV) (2.2 g) and dry triethylamine (1.70 g) in dry ethanol(20 ml) was refluxed for 2 hours. The mixture was cooled, water (50 ml)was added, and the resulting precipitate was filtered off and dried.Recrystallisation from a mixture of water and ethanol gave thenitropyrimidine oxime (II) (1.4 g, yield 48%) as pale yellow crystals,m.p. 242° (decomp.).

v.2-Amino-4-hydroxy-6-(p-methoxy)phenoxymethyl-7,7-dimethyl-7,8-dihydropteridine(I; R = CH₂ OC₆ H₄.OCH₃, R¹ = R² = CH₃)

A solution of2-amino-4-hydroxy-6[2-hydroxyimino-3-(p-methoxy)phenoxy-1,1-dimethyl-propylamino]-5-nitropyrimidine (II) (0.5 g) in water (15 ml) and 2M sodium hydroxide(3 ml) was warmed on a steam-bath. Sodium dithionite (1.0 g) was addedportionwise until an almost colourless solution was obtained. Glacialacetic acid was added to the solution to adjust to pH4, and the mixturewas heated for 1 hour. The solution was cooled and the solid (0.250 g)was filtered off. The solid was heated in dimethylformamide (40 ml) for2 hours on the steam bath. Filtration of the cooled reaction mixturegave the dihydropteridine (I) (0.18 g, yield 40%) m.p. 274°-276°(decomp.).

EXAMPLE D2-Amino-4-hydroxy-6-(p-chloro)phenoxymethyl-7,7-dimethyl-7,8-dihydropteridine(I; R = CH₂ OC₆ H₄.Cl, R¹ =R² =CH₃) i.1-(p-chloro)phenoxy-3-methylbut-2-ene (VI; R = CH₂ OC₆ H₄.Cl, R¹ = R² =CH₃)

This compound was prepared following the general procedures describedunder Example B(i), using p-chlorophenol as starting material instead ofp-methoxyphenol.

ii. 2-Chloro-4-(p-chloro)phenoxy-2-methyl-3-nitrosobutane (V; R = CH₂OC₆ H₄.Cl, R¹ = R² = CH₃)

Concentrated hydrochloric acid (28.4 ml) was added dropwise over 1 hourto a mixture of 1-(p-chloro)phenoxy-3-methylbut-2-ene (VI; R=CH₂ OC₆H₄.Cl, R¹ = R² = CH₃) (56.1 g) and amyl nitrite (42.6 ml) in glacialacetic acid (56.7 ml) at -5° to 0° (ice-salt bath). After a further 1hour at -5° to 0°, the mixture was filtered. The solid so obtained waswashed with benzene and recrystallised from benzene after drying to givethe nitrosochloride (V) (35.31 g, yield 47%) as pale yellow crystals,m.p. 124°-5° (decomp.).

iii. 3-Amino-1-(p-chloro)phenoxy-3-methylbutan-2-one oxime hydrochloride(IV; R = CH₂ OC₆ H₄.Cl, R¹ = R² = CH₃)

A suspension of 2-chloro-4-(p-chloro)phenoxy-2-methyl-3-nitrosobutane(V) (34.20 g) in dry methanol (375 ml) was heated to reflux whileammonia gas was passed through until all the solid dissolved (9.5hours). Filtration and removal of solvents gave a brown solid, which waslargely freed of impurities by washing with benzene. Recrystallisationfrom isopropanol (a small inorganic residue remained) gave the ketoximehydrochloride (IV) (19.65 g, yield 54%) as colourless crystals, m.p.225°-227° (decomp.).

iv.2-Amino-4-hydroxy-6-[2-hydroxyiminio-3-(p-chloro)phenoxy-1,1-dimethylpropylamino]-5-nitropyrimidine(II; R = CH₂ OC₆ H₄.Cl, R¹ = R² = CH₃)

A mixture of 3-amino-1-(p-chloro)-phenoxy-3-methylbutan-2-one oximehydrochloride (IV) (5.58 g),2-amino-4-chloro-6-hydroxy-5-nitropyrimidine (III) (Hal = Cl) (3.81 g)and dry triethylamine (8.2 g) was heated to reflux in dry ethanol (240ml) for 16 hours and filtered hot. The filtrate was reduced in volumeand the solid so obtained was filtered off. Recrystallisation fromethanol gave the nitropyrimidine oxime (II) (1.17 g) as a pale yellowgelatinous solid, m.p. >250°C (decomp.).

v. 2-Amino-4-hydroxy-6-(p-chloro)phenoxymethyl-7,7-dimethyl pteridine(I; R = CH₂ OC₆ H₄.Cl, R¹ = R² = CH₃)

A solution of2-amino-4-hydroxy-6-[2-hydroxyimino-1,1-dimethylpropylamino]-5-nitropyrimidine(II) in warm 1M sodium hydroxide was treated with sodium dithionite inportions and the title compound was isolated in analogous manner to theprocedure described under Example B(v).

EXAMPLE E2-Amino-4-hydroxy-6-(p-methyl)phenoxymethyl-7,7-dimethyl-7,8-dihydropteridine(I; R = CH₂ OC₆ H₄.CH₃, R¹ = R² = CH₃) i.1-(p-methyl)phenoxy-3-methylbut-2-ene (VI; R = CH₂ OC₆ H₄.CH₃, R¹ = R² =CH₃)

A mixture of p-cresol and sodium hydroxide was stirred withdimethylformamide and water while 1-bromo-3-methylbut-2-ene (VII; R¹ =R² = CH₃, Hal = Br) was added dropwise. The1-(p-methylphenoxy-3-methylbut-2-ene was isolated as described underExample B(i).

ii. 2-Chloro-4-(p-methyl)phenoxy-2-methyl-3-nitrosobutane (V; R = CH₂OC₆ H₄.CH₃, R¹ = R² = CH₃)

Concentrated hydrochloric acid (7.8 ml) was added dropwise over 1.5 hourto a mixture of 1-(p-methyl)phenoxy-3-methylbut-2-ene (VI; R = CH₂ OC₆H₄.CH₃, R¹ = R² = CH₃) (12.85 g) and amyl nitrite (11.7 ml) in glacialacetic acid (15.6 ml) at -5° to 0° (ice-salt bath). After a further 1hour at -5° to 0°, the mixture was filtered. The yellow solid soobtained was washed with acetone and recrystallised from acetone to givethe nitrosochloride (V) (7.5 g, yield 42%) as white crystals, m.p.144°-5° (decomp.).

iii. 3-Amino-1-(p-methyl)phenoxy-3-methylbutan-2-one oxime hydrochloride(IV; R = CH₂ OC₆ H₄.CH₃, R¹ = R² = CH₃)

A suspension of 2-chloro-4-(p-methyl)phenoxy-2-methyl-3-nitrosobutane(V) in dry methanol (120 ml) was heated to reflux and ammonia gas waspassed through until all the solid dissolved (2 hours). Filtration andevaporation of solvent gave a brown solid, which was largely freed ofimpurities by washing with benzene. Recrystallisation from isopropanol(a small inorganic residue remained) gave the ketoxime hydrochloride(IV) (5.27 g, yield 66%) as colourless crystals, m.p. 217°-8° (decomp.).

iv.2-Amino-4-hydroxy-6-(2-hydroxyimino-3-(p-methyl)phenoxy-1,1-dimethylpropylamino)-5-nitropyrimidine(II; R = CH₂ OC₆ H₄.CH₃, R¹ = R² = CH₃)

A mixture of 3-amino-1-(p-methyl)phenoxy-3-methylbutan-2-one oximehydrochloride (IV), 2-amino-4-chloro-6-hydroxy-5-nitropyrimidine (III)(Hal = Cl) (1.91 g) and dry triethylamine (4.08 g) was heated to refluxin dry ethanol (120 ml) for 16 hours. The resulting suspension wascooled and filtered.

The solid so obtained was partially dissolved in ethanol (500 ml) atreflux. On cooling, the filtered solution deposited the nitropyrimidineoxime (II) as a white gelatinous solid (0.87 g), m.p. >300° (decomp.).

v.2-Amino-4-hydroxy-6-(p-methyl)phenoxymethyl-7,7-dimethyl-7,8-dihydropteridine(I; R=CH₂ OC₆ H₄.CH₃, R¹ =R² =CH₃)

A solution of2-amino-4-hydroxy-6-[2-hydroxyimino-3-(p-methyl)phenoxy-1,1-dimethylpropylamino]-5-nitropyrimidine(II) (1.2 g) in warm 1 M sodium hydroxide (50 ml) was treated during 10minutes with sodium dithionite (5 g) until the initial red colour wasreplaced by a white solid. After 2 hours on a steam bath the suspensionwas cooled and filtered and the dihydropteridine (I) was precipitatedout. It was purified by dissolving it in dimethylsulphoxide, filteringthe resulting solution and diluting with water to precipitate theproduct as a white powder, m.p. >250° (decomp.).

EXAMPLE F2-Amino-4-hydroxy-6-(p-methoxy)phenoxymethyl-7,7-diethyl-7,8-dihydropteridine(I; R = CH₂ OC₆ H₄.OCH₃, R¹ = R² = C₂ H₅) i.1-(p-methoxy)phenoxy-3-ethylpent-2-ene (VI; R = CH₂ OC₆ H₄.OCH₃, R¹ = R²= C₂ H₅)

A solution of p-methoxyphenol (24.5 g) and sodium hydroxide pellets (9g) in dimethylformamide (200 ml) and water (20 ml) at 60° was treatedwith 1-bromo-3-ethyl-2-pentene (VII), (R¹ = R² = C₂ H₅) (35 g), addeddropwise with stirring over 1-11/2 hours. The reaction mixture wasstirred for a further 4 hours at 60°-65° and then poured into ice-water(750 ml). The mixture was extracted with ether (3×300 ml) and theextract was washed with 10% sodium hydroxide solution (200 ml), water(3×200 ml) and brine (50 ml). It was dried over sodium sulphate.Evaporation of the ether gave a reddish-brown oil which on distillationgave the product (26 g), b.p. 90°-95°/0.2 torr.

ii. 3-Chloro-1-(p-methoxy)phenoxy-3-ethyl-2-nitrosopentane (V; R = CH₂OC₆ H₄.OCH₃, R¹ = R² = C₂ H₅)

1-(p-methoxy)phenoxy-3-ethyl-pent-2-ene (26 g) (VI) in glacial aceticacid (25 ml) and amyl nitrite (17.7 ml) at 0° was treated withconcentrated hydrochloric acid (11.8 ml) added dropwise with stirringover 1 hour. The reaction mixture was stirred for a further 1 hour andthe yellow precipitate which had formed was filtered and dried on aporous plate (12.2 g). A portion (1 g) was recrystallised from acetoneto give a pale yellow crystalline solid, m.p. 122°-3°.

iii. 3-Amino-1-(p-methoxy)phenoxy-3-ethylpenten-2-one oximehydrochloride (IV; R = CH₂ OC₆ H₄.OCH₃, R¹ = R² = C₂ H₅)

3-Chloro-1-(p-methoxy)phenoxy-3-ethyl-2-nitrosopentane (V) (11.2 g) wassuspended in methanolic-ammonia solution (250 ml) and refluxed whilstammonia was bubbled through the solution. After 6 hours themethanol-ammonia was removed in vacuo to give a brown gum. Anhydrousbenzene (50 ml) was added to dissolve the gum and the addition ofpetroleum ether 40°-60° (100 ml) precipitated an off-white crystallinesolid. This was dissolved in boiling sec-butanol (200 ml), filtered andthe filtrate concentrated to a small volume (˜50 ml) to yield acrystalline solid (4.3 g), m.p. 190°(dec.).

Concentration of the mother liquors gave a second crop of product (1.7g).

iv.2-Amino-4-hydroxy-6-(2-hydroxyimino-3-(p-methoxy)phenoxy-1,1-diethylpropylamino)5-nitropyrimidine(II; R = CH₂ OC₆ H₄.OCH₃, R¹ = R² = C₂ H₅)

A mixture of 3-amino-1-(p-methoxy)phenoxy-3-ethylpenten-2-one oximehydrochloride (IV) (4.8 g), 2-amino-4-chloro-6-hydroxy-5-nitropyrimidine(III) (Hal=Cl) (3 g) and triethylamine (5 g) in absolute ethanol (200ml) was refluxed for 18 hours. The reaction mixture was cooled and theinsoluble residue filtered. The filtrate was evaporated to dryness invacuo to give a pale yellow gum. This was taken up in hot acetone (100ml) and the residue filtered. The filtrate was concentrated in vacuo to30 ml and treated with water (150 ml) to precipitate a gum whichsolidified on refrigeration. This was filtered and dried in vacuo (3.6g). A portion (0.6 g) was recrystallised from aqueous acetone (in thepresence of charcoal) to give an off-white crystalline solid,m.p. >250°. The bulk was taken up in acetone (50 ml), filtered to removeinsoluble residue (1 g) and the product was precipitated by the additionof water (50 ml) to give pure product (1.6 g).

v.2-Amino-4-hydroxy-6-(p-methoxy)phenoxymethyl-7,7-diethyl-7,8-dihydropteridine(I; R = CH₂ OC₆ H₄.OCH₃ R¹ = R² = C₂ H₅)

2-Amino-4-hydroxy-6-[2-hydroxyimino-3-(p-methoxy)phenoxy-1,1-diethylpropylamino]-5-nitropyrimidine(II) (0.8 g) in 2N sodium hydroxide (100 ml) was treated with sodiumdithionite whilst heating on steambath. The solution turned areddish-brown and then to pale yellow-green. The reaction mixture wasleft on a steambath for 45 minutes, then cooled and taken to pH8 withglacial acetic acid to precipitate a colourless flocculent solid. Thiswas filtered to give a tackycolourless solid (0.3 g). Recrystallisationfrom aqueous alcohol gave an off-white solid (0.08 g).

EXAMPLE G2-Amino-4-hydroxy-6-(p-chloro)phenoxymethyl-7,7-diethyl-7,8-dihydropteridine(I; R = CH₂ OC₆ H₄.CL, R¹ = R² = C₂ H₅) i.1-(p-chloro)phenoxy3-ethylpent-2-ene (VI; R = CH₂ OC₆ H₄.Cl, R¹ = R² =C₂ H₅)

A solution of p-chloro-phenol (26.5 g) and sodium hydroxide pellets (9g) in dimethylformamide (180 ml) and water (25 ml) at 60° was treatedwith 1-bromo-3-ethyl-2-pentene (VIII), (R¹ = R² = C₂ H₅) (36.5 g), addeddropwise with stirring over 1-11/2 hours. The reaction mixture wasstirred for a further 4 hours at 60°-65° and then poured into ice-water(750 ml). The mixture was extracted with ether (3 × 300 ml) and theextract was washed with 10% sodium hydroxide solution (200 ml), water (3× 200 ml) and brine (50 ml).

It was dried over sodium sulphate. The ether was removed in vacuo togive a pale brown oil (36 g) which on distillation gave the product(32.5 g), b.p. 112°-15°/0.6 torr.

ii. 3-Chloro-1-(p-chloro)phenoxy-3-ethyl-2-nitrosopentane (V; R = CH₂OC₆ H₄.Cl, R¹ = R² = C₂ H₅)

1-(p-chloro)phenoxy3-ethylpent-2-ene (VI) (32.5 g) in glacial aceticacid (29 ml) and amyl nitrite (21.6 ml) at 0° was treated withconcentrated hydrochloric acid (14.5 ml) added dropwise with stirringover 1 hour. The reaction mixture was stirred at 0° for a further 1 hourand the yellow precipitate which had formed was filtered and dried on aporous plate (19.5 g). It was recrystallised from acetone to give anoff-white crystalline solid, (15 g), m.p. 129° (dec.)

iii. 3-Amino-1-(p-chloro)phenoxy-3-ethyl-penten-2-one oximehydrochloride (IV; R = CH₂ OC₆ H₄.Cl, R¹ = R² = C₂ H₅)

3-Chloro-1-(p-chloro)phenoxy-3-ethyl-2-nitrosopentane (V) (15 g) wassuspended in methanolic ammonia solution (300 ml) and refluxed whileammonia was bubbled through the solution. After 7 hours all the startingmaterial had dissolved and the solution was cooled and themethanol-ammonia evaporated in vacuo to give a colourless tacky solid.This was dissolved up in sec-butanol (250 ml) with heating on asteambath, filtered to remove a small amount of insoluble residue andthe filtrate concentrated until solid began to be deposited. It wasrefrigerated overnight to give a crop of colourless crystals (9.2 g).Evaporation of the mother liquors gave a second crop of product (1.5 g).m.p.>200°.

iv.2-Amino-4-hydroxy-6-(2-hydroxyimino-3-(p-chloro)phenoxy-1,1-diethylpropylamino)-5-nitropyrimidine(II: R = CH₂ OC₆ H₄.Cl, R¹ = R² = C₂ H₅)

A mixture of 3-amino-1-(p-chloro)phenoxy-3-ethylpenten-2-one oximehydrochloride (IV) (5 g), 2-amino-4-chloro-6-hydroxy-5-nitropyrimidine(III) (Hal=Cl) (3 g) and triethylamine (5 g) in absolute ethanol (200ml) was refluxed together for 18 hours. The reaction mixture was cooledand filtered to remove the solid residue (1 g). The filtrate wasevaporated in vacuo to dryness to give a pale yellow oil which was thentreated with water (150 ml) to precipitate a yellow-brown oil whichsolidified to give a tacky solid. This was dissolved in hot acetone (80ml) and the residue filtered. Evaporation of the filtrate gave a gumwhich rapidly solidified (2.5 g). A portion (0.5 g) was recrystallisedfrom aqueous acetone to give pale yellow needles, m.p. 217° (dec.). Theremainder was recrystallised to give pure product (as judged by t.l.c.)(1.4 g).

v.2-Amino-4-hydroxy-6-(p-chloro)phenoxymethyl-7,7-diethyl-7,8-dihydropteridine(I; R = CH₂ OC₆ H₄.Cl, R¹ = R² = C₂ H₅)

2-Amino-4-hydroxy-6-(2-hydroxyimino-3-(p-chloro)phenoxy-1,1-diethylpropylamino)-5-nitropyrimidine (0.5 g) in 2N sodium hydroxide(80 ml) was treated with sodium dithionite with heating on thesteambath. The solution turned a reddish-brown and then to a paleyellow. The reaction mixture was left for 45 minutes on a steambath andthen cooled and taken to pH8 with glacial acetic acid, whereupon thedihydropteridine separated as an off-white solid (0.05 g).

EXAMPLE H 2-Amino-4-hydroxy-6-(p-methyl)phenoxymethyl7,7-diethyl-7,8-dihydropteridine (I; R = CH₂ OC₆ H₄.CH₃, R¹ = R² = C₂ H₅i. 1-(p-methyl)phenoxy-3-ethylpent-2-ene (VI; R = CH₂ OC₆ H₄.CH₃, R¹ =R² = C₂ H₅)

A solution of p-cresol (20.8 g) and sodium hydroxide pellets (7.7 g) indimethylformamide (150 ml) and water (20 ml) at 60°-70° was treated with1-bromo-3-ethyl-2-pentene (VII) (R¹ = R² = C₂ H₅), (34 g), addeddropwise with stirring over 1-11/2 hours. The mixture was stirred for afurther 4 hours at 60°-65° and then poured into ice-water (750 ml). Themixture was extracted with ether (3 × 300 ml) and the extract was washedwith 10% sodium hydroxide (200 ml), water (3 × 200 ml) and brine (50ml). It was dried with sodium sulphate. Evaporation of the ethersolution gave a pale yellow oil (26.6g) which on distillation gave theproduct (18 g), b.p. 99°-102°/0.35 torr.

ii. 3-Chloro-1-(p-methyl)phenoxy-3-ethyl-2-nitrosopentane (V; R = CH₂OC₆ H₄.CH₃, R¹ = R² = C₂ H₅)

1-(p-methyl)phenoxy-3-ethylpent-2-ene (VI) (18 g) in glacial acetic acid(17.6 ml) and amyl nitrite (13.2 ml) at 0° was treated with concentratedhydrochloric acid (8.8 ml) added dropwise over 1 hour. The reactionmixture was stirred at 0° for a further 1 hour and the plate yellowsolid which precipitated was filtered and dried on a porous plate (14.5g). A portion (1 g) was recrystallised from acetone to give a paleyellow crystalline solid, m.p. 129° (dec.).

iii. 3-Amino-1-(p-methyl)phenoxy-3-ethylpenten-2-one oxime hydrochloride(IV; R = CH₂ OC₆ H₄.CH₃, R¹ = R² = C₂ H₅)

3-Chloro-1-(p-methyl)phenoxy-3-ethyl-2-nitrosopentane (13.8 g) wassuspended in methanolic-ammonia solution (250 ml) and refluxed whilstammonia was bubbled through the solution. After 6 hours themethanol-ammonia was removed in vacuo and the residue was treated withacetone (50 ml) to precipitate the crude ketoxime hydrochloride (7 g).This was treated with benzene (50 ml) and the residue was recrystallisedfrom isopropanol to give a colourless crystalline solid (4.8 g).

iv.2-Amino-4-hydroxy-6-(2-hydroxyimino-3-(p-methyl)phenoxy-1,1-diethylpropylamino)-5-nitropyrimidine(II; R = CH₂ OC₆ H₄.CH₃, R¹ = R² = C₂ H₅)

3-Amino-1-(p-methyl)phenoxy-3-ethyl-penten-2-one oxime hydrochloride(IV), 2-amino-4-chloro-6-hydroxy-5-nitropyrimidine (III) (Hal=Cl) andtriethylamine in absolute ethanol were refluxed together for 18 hours.The reaction mixture was cooled and the insoluble residue removed byfiltration. The filtrate was evaporated in vacuo to dryness and thetitle compound was isolated analogously to the procedure described underExample F (iv).

2-Amino-4-hydroxy-6-(p-methyl)phenoxymethyl-7,7-diethyl-7,8-dihydropteridine(I; R = CH₂ OC₆ H₄,CH.sub. 3, R¹ = R² = C₂ H₅)

2-Amino-4-hydroxy-6-(2-hydroxyimino-3-(p-methyl)phenoxy-1,1-diethyl-propylamino)-5-nitropyrimidine (II) in 2N sodium hydroxide wastreated with sodium dithionite whilst heating on a steambath. The titlecompound was isolated analogously to the procedure described underExample F (v).

EXAMPLE I Starting Material for Examples A - E i. 3-Methyl-2-buten-1-ol(VIII); R¹ =R² =CH₃)

A solution of 3,3-dimethylacrylic acid (IX; R^(1=R) ² =CH₃, Y=H) (20 g)in sodium-dried ether (200 ml) was treated dropwise with 64 g of a 70%solution of sodium dihydro-bis(2-methoxyethoxy) aluminate in benzene,the temperature being maintained at 0° until the addition was complete(about 1h). The reaction mixture was stirred for 5h at room temperature,and water was then added slowly. The sodium aluminate which precipitatedwas filtered off, and the filtrate was extracted with ether (4 × 100ml). The combined extracts were dried, the solvent was removed, and theresidual oil was distilled to give 3-methyl-2-buten-1-ol (VIII) as acolourless liquid, b.p. 54°-55°/25mm Hg.

ii. 1-Bromo-3-methylbut-2-ene (VII, R¹ =R² =CH₃, Hal=Br)

3-methyl-2-buten-1-ol (VIII) in dry ether was reacted with phosphorustribromide following the procedure described below in Example J (ii) toproduce 1-bromo-3-methylbut-2-ene (VII), which could be used directlyfor the preparation of the corresponding compounds of formula (VI).

EXAMPLE J Starting Material for Examples F - H i. 3-Ethyl-1-penten-3-ol(X; R^(1=R) ² =C₂ H₅)

To a solution of vinylmagnesium bromide prepared from 29.2 g magnesiumwas added a solution of penten-3-one (XI; in =R² =C₂ H₅) (76 g) intetrahydrofuran (80 ml) keeping the temperature below 50°. After theaddition was complete the reaction mixture was left for 15 h at roomtemperature, then cooled to 0° and diluted with water (500 ml), addedcautiously with vigorous stirring keeping the temperature below 40°. Theorganic phase was separated and the aqueous phase back-extracted withether. The extracts were combined and dried with magnesium sulphate. Thesolvent was removed i n vacuo and the residue was distilled to give3-ethyl-1-penten-3-ol (60 g) b.p. 131°-2°.

ii. 1-Bromo-3-ethyl-2-pentene (VII; R¹ =R² =C₂ H₅, Hal=Br)

3-Ethyl-1-penten-3-ol (X) (25 g) in dry ether (200 ml) was stirred at-10° to -2° under nitrogen. Phosphorus tribromide (35 g) was addeddropwise over about 30 minutes and the mixture for a further 3 hours at-10° to 0°, before pouring into ice-water (200 ml). The ethereal layerwas separated and the aqueous layer back-extracted with ether (150 ml).The combined extracts were then washed with water, saturated sodiumbicarbonate and saturated sodium chloride solutions and then dried withmagnesium sulphate. The ether was removed in vacuo to give a colourlessoil (34 g) which was not distilled but used directly for the preparationof the compounds of formula (VI).

The same product could be obtained using 3-ethyl-2-penten-1-ol (VIII; R¹=R² =C₂ H₅) as the starting material for this stage.

EXAMPLE K

Potential pteridine antagonists of formula (I) may be tested byinvestigating the inhibitory effect they impose on the enzymesresponsible for the biosynthesis of dihydropteroic acid (DPtA), namelyhydroxymethyldihydropteridine pyrophosphokinase (HMPPS), anddihydropteroate synthetase, hereinafter referred to as `synthetase`. Inthe following reaction equations the compounds are referred to by theirabbreviated forms defined on page 5 of the specification.

    ______________________________________                                        1. HMPPS                                                                      Mg.sup.2.sup.+                                                                HMPt + ATP ⃡Pt + AMP                                              2. Synthetase                                                                 Mg.sup.2.sup.+                                                                Pt + pAB ⃡DPtA + pyrophosphate                                    ______________________________________                                    

a. An assay for HMPPS was developed in which the transfer of theterminal phosphate of ATP-γ-P³² to Pt could be monitored and correlatedwith the amount of inhibition of HMPPS by the compound under test.

The compound of formula (I) which was under test was incorporated intovarious formulations comprising metabolites and enzymes contained intest tubes, as indicated in TABLE 1.

The components of the mixture were as follows:

I--2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine (HMPt) in aconcentration of 800 μM i.e. micromolar;

II--a source of HMPPS, obtained from an extract of E. coli and separatedfrom `synthetase` on Sephadex G-100, (Registered Trade Mark) accordingto the method of Richey and Brown in J. Biol. Chem. 244, 1582-1592(1969).

III--3mM ATP -γ-P³².

IV--0.10 M ATP neutralised (unlabelled).

V--0.02M MgCl₂.6H₂ O.

VI--0.1 M MgCl₂.6H₂ O.

VII--Source of HMPPS and `synthetase`.

VIII--the test compound in a concentration of 0.93×10⁺ ³ M IX--0.4mMpAB-C¹⁴

As shown in TABLE 1, tubes 1 to 9 all contain a source of HMPPS,labelled ATP and 0.02 M MgCl₂.6H₂ O, tubes 2 to 9 containing in additionHMPt and tubes 4 to 9 further containing the test compound. Controltubes 10 to 12 include a source of both HMPPS and synthetase, unlabelledATP, 0.1M MgCl₂.6H₂ O and labelled pAB.

Tubes 1 to 9 containing the amounts of components shown in the Table,were filled up to 200μ1 with distilled water, incubated for 60 minutesat 37°C and then chilled on ice. Dextrose (20μ1 containing 72.1 mg/ml)and hexokinase (5μ1 containing 2000 units/ml) were added to thesolution, which was then allowed to stand at room temperature for 15minutes. `Darco-G-60` (Registered Trade Mark) (10 mg) was added to eachtube and the contents mixed periodically for 10 minutes. The charcoalwas removed through a `Millipore AP 250 2200` (Registered Trade Mark)filter and the filter was washed with three 10 ml portions of coldwater. The charcoal and the filter were then radioactively counted.

The radioactive count from the contents of tubes 2 and 3 was taken asthe maximum count, since these tubes contained no test compound and thusgave 0% enzyme inhibition. The percentage inhibition produced by thecontents of the remaining tubes could then be calculated by relatingtheir radioactive count to the maximum, as determined above.

The contents of tubes 10 to 12 were chromatographically analysed asdescribed under part (b), and used as controls, tubes 10 and 11containing no test compound (and hence giving 0% inhibition) beingaccorded the value of 100%. The percentage inhibition exhibited by thecontents of the tubes in part (b) of the experiment could then becalculated in relation to this, by comparing the respectivechromatograms.

b. The activity of the test compound of formula (I) against `synthetase`was determined as follows, by monitoring the formation ofdihydropteroate C¹⁴.

A pool of Pt was prepared from ATP neutralised (50μl l, 0.1M), MgCl₂.6H₂O (50μl,0.1M), dithiothreitol (100μl,0.1M), tris buffer (100μl,0.4M,pH8.3), HMPt (25μl,876μM) and 170μl of a solution containing HMPPS. Themixture was incubated for 60 minutes at 37°C, chilled briefly on ice andthen dextrose (100μl containing 72.1mg/ml) and hexokinase (20μlcontaining 2000 units/ml) were added at room temperature to thesolution, which was allowed to stand at this temperature for 15 minutes.

A solution of MgCl₂.6H₂ O (10μ1,0.1M), pAB-C¹⁴ (10μl,0.4mM),dithiothreitol (20μl,0.1M) and tris buffer (20μl, 0.4M,pH8.3) was madein each of five test tubes and then 80μl of the contents of the pooladded to each, together with synthetase and/or test compound of formula(I) as indicated in Table 2. The solution was then made up to 200μl withdistilled water.

Two control test tubes were prepared, each containing ATP (10μl,0.1M),MgCl₂.6H₂ O(10μl,0.1M), dithiothreitol (20μl,0.1M) tris buffer(20μl,0.4M,pH 8.3), pAB-C¹⁴ (10μ1, 0.4mM), and 20μl of a solutioncontaining HMPPS and `synthetase` of known activity. The test compoundwas added to the second of these two tubes up to a final concentrationof 10⁺ ⁵ M, and both tubes were made up with distilled water to 200μl.

All seven tubes were then incubated for 30 minutes at 37°C, chilled onice and then these, together with control tubes 10 to 12 from part (a),were chromatographically analysed as follows.

100μl of the contents of each of the tubes was spotted onto Whatman no.3MM chromatography paper (2×20 cm) at the `origin`, the run descendingin a Srenson buffer of potassium and sodium phosphates (0.1M,pH 7.0) for10 to 15 cm. From the relative positions of the spots obtained from thecontents of the different tubes, the various percentage inhibitions ofsynthetase could be evaluated by reference to control tubes 10 and 11,which gave 0% inhibition.

Those compounds which, as result of these tests, were found to give 50%inhibition at a concentration of 100μM or less, are those which exert auseful potentiating effect, and subject to their toxicity beingfavourable, may be included in the compositions described in thisspecification.

2-Amino-4-hydroxy-6-phenoxymethyl-7,7-dimethyl-7,8-dihydropteridine wasfound to give 50% inhibition at a concentration of 17 μM.

EXAMPLE L

    Tablet Formulation                                                            Compound of formula (I)(R=CH.sub.2 Ph;R.sup.1 =R.sup.2 =Me)                                                100 mg                                           Trimethoprim (pure)           25 mg                                           Sulfaguanidine (B.P.C.)      100 mg                                           + cornstarch, lactose, gelatin, talcum and                                    magnesium stearate                                                        

Preparation -- the above constituents were mixed together using knownmethods of pharmacy to form a granulation which was then compressed intotablets.

EXAMPLE MTablet Formulation"Pyremathimine" (Pyrimethamine) B.P. 15mgCompound of formula (I)(R=CH₂ Ph;R¹ =R² =Me) 150 mg

which was then prepared to form a tablet as in Example L.

Example N

    Tablet Formulation                                                            Sulfanilamide B.P.C.         150 mg                                           Compound of formula (I)(R=CH.sub.2 Ph;R.sup.1 =R.sup.2 =Me)                                                175 mg                                       

which was then prepared to form a tablet as in Example L.

EXAMPLE O

    Capsule Formulation                                                           Trimethoprim (pure)           20mg                                            Compound of formula (I)(R=CH.sub.2 Ph;R.sup.1 =R.sup.2 =Me)                                                100 mg                                       

Preparation -- The compounds in granular form were blended together withlactose, cornstarch and magnesium stearate. The powder was filled into atwo-piece, hard shell gelatin capsule using a capsulating machine.

EXAMPLE P

    Irrigant Solution                                                             Compound of formula (I) (R=CH.sub.2 Ph;R.sup.1 =R.sup.2 =Me)                                              1 mg/ml                                           (pure)                                                                        Trimethoprim (pure)        0.2mg/ml                                           Solvent                    water                                          

EXAMPLE Q

    Irrigant Solution                                                             Compound of formula (I) (R=CH.sub.2 Ph;R.sup.1 =R.sup.2 =Me)                                               2mg/ml                                           (pure)                                                                        amino-p-toluenesulphonamide (pure)                                                                         2mg/ml                                       

EXAMPLE R

    Solution                                                                      Compound of formula (I) (R=CH.sub.2 Ph;R.sup.1 =R.sup.2 =Me)                                             1.5 mg/ml                                          (pure)                                                                        Diaveridine B. Vet C       0.5 mg/ml                                          Kelfizina                  1.0 mg/ml                                          Solvent                    water                                          

EXAMPLE S

    Tablet Formulation                                                            Compound of formula (I) (R=CH.sub.2 Ph;R.sup.1 =R.sup.2 =Me)                                             500 mg                                             (pure)                                                                        Microcrystalline cellulose 100 mg                                             Starch                     40 mg                                              Magnesium stearate         10 mg                                              Methylhydroxyethylcellulose                                                                              3 mg                                                                          653 mg                                         

The pteridine (I), microcrystalline cellulose and starch were granulatedwith a solution of the methylhydroxyethylcellulose in 50% aqueous ethylalcohol. The magnesium stearate was added to the dried granules, and thewhole then compressed.

                                      TABLE 1.                                    __________________________________________________________________________    Tube No.                                                                            I   II   III IV  V   VI  VII VIII   IX                                                                     Final                                                                         Concn.                                     __________________________________________________________________________    1     --  100μl                                                                           15μl                                                                           --  10μl                                                                           --  --  --     --                                  2     5μl                                                                            "    "   --  "   --  --  --     --                                  3     "   "    "   --  "   --  --  --                                         4     "   "    "   --  "   --  --  2.5×10.sup..sup.-6 M                 5     "   "    "   --  "   --  --  "                                          6     "   "    "   --  "   --  --  1.0×10.sup..sup.-5 M                                                           --                                  7     "   "    "   --  "   --  --  "      --                                  8     "   "    "   --  "   --  --  9.3×10.sup..sup.-5 M                                                           --                                  9     "   "    "   --  "   --  --  "      --                                  Controls                                                                      10    --  --   --  10μl                                                                           --  10μl                                                                           20μl    10μl                             11    5μl                                                                            --   --  "   --  "   "          "                                   12    "   --   --  "   --  "   "   1.0×10.sup..sup.-5 M                                                           "                                   __________________________________________________________________________

                  TABLE 2                                                         ______________________________________                                        Tube No.     Excess         Test compound                                                  Synthetase     Final Concen-                                                                 tration.                                          ______________________________________                                        1            -              --                                                2            +              --                                                3            +              8.7 × 10.sup..sup.-5 M                      4            +              1.0 × 10.sup..sup.-5 M                      5            +              2.5 × 10.sup..sup.-6 M                      Controls                                                                      6            -              --                                                --           -              1.0 × 10.sup..sup.-5 M                      ______________________________________                                    

What we claim is:
 1. A compound of the formula ##SPC5##wherein R isphenoxyloweralkyl or phenoxy lower alkyl which is substituted by one ormore lower alkoxy, amino, lower alkyl, hydroxyl or halogen and R¹ and R²are the same or different and each is lower alkyl or R¹ and R², havingin total 4 to 6 carbon atoms, form a spirocycloalkyl group together withthe carbon atom substituted by said R¹ and R² Z is an oxygen atom or aprotecting group therefore, or a tautomeric form thereof, and wherein inthe above lower alkyl and lower alkoxy have 1 to 4 carbon atoms.
 2. Thecompound according to claim 1 in which there is a para substitution inthe phenyl ring.
 3. The compound according to claim 1 in whichphenoxyalkyl is substituted with methyl, methoxy, or chlorine.
 4. Thecompound according to claim 1 in which R is phenoxymethyl.
 5. Thecompound according to claim 1 in which R¹ and R² are methyl.
 6. Thecompound according to claim 1 in which the protecting group is oxime orsemicarbazone.
 7. The compound according to claim 5 in which Z isoxygen, oxime or semicarbazone.
 8. The compound according to claim 5 inwhich Z is oxime.
 9. The compound according to claim 1 in which R¹ andR² are CH₃, Z is oxime and R is CH₂ OC₆ H₄.OCH₃.
 10. The compoundaccording to claim 1 in which R¹ and R² are CH₃, Z is oxime and R is CH₂OC₆ H₄.Cl.
 11. The compound of claim 5 in which R¹ and R² are CH₃, Z isoxime and R is CH₂ OC₆ H₄.CH₃.
 12. The compound2-Amino-4-hydroxy-6-[2-hydroxyimino-3-(p-methoxy)phenoxy-1,1-dimethylpropylamino]-5-nitropyrimidine.